Human ADRB1 Stable Cell Line - HEK293

Multiple intracellular residues of G protein-coupled receptors (GPCRs) are phosphorylated by G protein-coupled receptor kinases. The resulting phosphorylation pattern triggers the recruitment of arrestin proteins and receptor desensitization. For the human β1-adrenergic receptor (ADRB1), the precise phosphorylation sites and their functions remain largely unknown. ADRB1 is a key GPCR in adrenergic signaling and a target for commonly used drugs such as beta-blockers. Here, researchers constructed human ADRB1-overexpressing HEK293 cells and determined the phosphorylation pattern of ADRB1 before and after receptor agonist stimulation using mass spectrometry. Eight phosphorylation sites were identified in the third intracellular loop and the C-terminus of the receptor. By analyzing the functional relevance of each site using receptor mutants lacking specific phosphorylation sites, the researchers found that phosphorylation at the Ser461/Ser462 sites in the distal C-terminus of ADRB1 determines the recruitment of β-arrestin 2 and receptor endocytosis. These data reveal the phosphorylation pattern of human ADRB1 and the key sites mediating β-arrestin 2 recruitment.

Immunoprecipitation of the ADRB1 from ADRB1-overexpressing HEK293 cells supplemented with ³²P and subsequent Western blotting lead to the detection of two specific bands for the ADRB1 at 55 and 68 kDa (Fig 1A, lower panel). Autoradiography of the blot revealed specific phosphorylation of ADRB1 (Figure 1A, upper panel). Compared to control cells, the phosphorylation level increased by 57 ± 5% after 5 minutes of stimulation with norepinephrine (NE, 100 μM) (Figure 1B). Subsequently, the phosphorylation pattern of ADRB1 in the basal state and after norepinephrine stimulation was analyzed using mass spectrometry (Figure 1C). ADRB1 was purified from ADRB1-overexpressing HEK293 cells using cross-linking immunoprecipitation. Subsequent SDS-PAGE and silver staining showed the two expected bands for ADRB1 (Figure 1D and 1E). Mass spectrometry analysis indicated that the low molecular weight band detected by silver staining in untransfected HEK293 cells also contained tubulin. The researchers also found that 8 out of 16 serine/threonine residues in the third intracellular loop and C-terminus were phosphorylated (Figure 1F). Under unstimulated conditions, Thr404 showed high phosphorylation levels, while Ser274, Ser412, and Ser461/Ser462 were mainly phosphorylated after norepinephrine stimulation.

Figure 1. The β₁-adrenoceptor is phosphorylated at multiple sites in the third intracellular loop and at the C-terminus. (Hinz L, et al., 2017)